Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal form of cancer that is expected to be the second leading cause of cancer death in the United States by 2020. One of the major factors that contribute to the poor prognoses of many PDAC patients is formation of inoperable metastases. Metastasis is a complex biological process that begins with malignant cells leaving the primary tumor and entering the bloodstream. In the latter part of this process, which is referred to as metastatic seeding, these cells exit the bloodstream, invade and colonize organs, and grow into secondary tumors at distant sites. Despite being a major determinant in the clinical outcomes of PDAC cases, the molecular and cellular events that drive metastatic seeding are largely unknown. This proposal will identify key proteins and/or pathways involved in metastatic seeding and provide insight into the molecular underpinnings of this process. We present a novel multiplexed in vivo screening platform using unique PDAC cell lines with `genetic barcodes', to discover small molecule inhibitors of metastatic seeding. Integral to the screening workflow is a focused library of compounds that target multiple classes of hydrolytic enzymes, which are known to be involved in metastatic seeding. Importantly, these molecules contain electrophilic traps to covalently modify the catalytic residues of target enzymes. Validated inhibitors of metastasis will be synthetically converted into activity-based probes that contain a `chemical handle', allowing us to isolate and identify covalently linked target-drug complexes by mass spectrometry. Identified targets will be validated by the generation of stable shRNA knockdown cell lines. These cell lines will be screened in the original in vivo metastatic seeding model, as well as secondary mouse models using intrasplenic injection and orthotopic transplantation to model the entire metastatic process. Overall, this proposal will validate a new approach for identifying small molecule inhibitors of metastatic seeding and use these compounds as chemical tools to uncover key targets involved in this process.